Arch Virol (2010) 155:1871–1876 DOI 10.1007/s00705-010-0773-2

BRIEF REPORT

A putative new ampelovirus associated with grapevine leafroll disease

N. Abou Ghanem-Sabanadzovic • S. Sabanadzovic • J. K. Uyemoto • D. Golino • A. Rowhani

Received: 29 March 2010 / Accepted: 24 July 2010 / Published online: 12 August 2010 Ó The Author(s) 2010. This article is published with open access at Springerlink.com

Abstract A putative new ampelovirus was detected in (GLRaVs) have been identified and characterized from Vitis vinifera cv. Carnelian showing mild leafroll symp- leafroll-affected grapevines [21]. Some of these viruses toms and molecularly characterized. The complete genome have been recognized as belonging to definitive species in consisted of 13,625 nt and had a structure similar to that of the genera Closterovirus (GLRaV-2) and Ampelovirus members of subgroup I in the genus Ampelovirus (fam. (GLRaV -1, -3 and -5) or unassigned species in the family Closteroviridae). In-depth analyses showed that the virus Closteroviridae (GLRaV-7) [22], whereas several viruses/ from cv. Carnelian is the most distinct member of the variants (i.e. GLRaV-4, -6, -9, -Pr, -De) still await official ‘‘GLRaV-4 lineage’’ of ampeloviruses, which comprises taxonomic assignment. The ampeloviruses associated with GLRaV-4, -5, -6, -9, and the recently characterized LRD are transmitted by grafting and by several species of GLRaV-Pr, and GLRaV-De. This virus appears to be a new mealybugs and soft scales [reviewed in ref. 23]. member of the family Closteroviridae, for which the pro- Mild downward rolling and premature reddening of the visional name grapevine leafroll-associated Carnelian virus leaves, resembling leafroll disease, were observed in 2005 is proposed. and 2006 on a grapevine (Vitis vinifera) cv. Carnelian [(Carignane 9 Cabernet Sauvignon) 9 Grenache] in a grape virus collection at the University of California, Davis. The Leafroll disease (LRD) is one of the most economically presence of leafroll infection was confirmed by grafting onto important and widespread viral diseases of cultivated the leafroll-specific indicator V. vinifera cv. Cabernet Franc grapevines. Several filamentous, phloem-restricted viruses (Fig. 1a). The original plant repeatedly tested negative in referred to as grapevine leafroll-associated viruses ELISA and RT-PCR for known grapevine leafroll-associ- ated viruses, thus justifying further investigation. The emphasis in this paper is given to a detailed comparison of The sequence reported in this paper is available in GenBank under this virus to other ‘‘GLRaV-4-like’’ viruses/variants. accession number FJ907331. Double-stranded RNA (dsRNA) was isolated from dis- eased tissues using double phenol–chloroform extractions & N. Abou Ghanem-Sabanadzovic D. Golino A. Rowhani ( ) and CF-11 column chromatography [6], purified by selective Department of Plant Pathology, University of California, Davis, CA 95616, USA enzymatic digestions [27] and analyzed by polyacrylamide e-mail: [email protected] gel electrophoresis (PAGE). The multiple high-molecular- weight dsRNA molecules in cortical tissue of the diseased & S. Sabanadzovic ( ) cv. Carnelian were interpreted to be replicative forms of a Department of Entomology and Plant Pathology, Mississippi State University, Mississippi State, clostero-like virus. The largest dsRNA molecule, interpreted MS 39762, USA as a full genomic-size replicon, migrated slightly faster than e-mail: [email protected] the corresponding molecule of grapevine leafroll-associated virus 2, indicating a smaller genome size. Based on differ- J. K. Uyemoto USDA-ARS, Department of Plant Pathology, ences in migration rates compared to standards (GLRaV-2 University of California, Davis, CA 95616, USA and grapevine rupestris stem pitting-associated virus, 123 1872 N. Abou Ghanem-Sabanadzovic et al.

sequencing multiple independent clones in order to achieve 12 3 at least 59 coverage/nucleotide. Uniform sequence data among multiple sequenced clones was further proof of the absence of related GLRaVs in the accession of cv. Carnelian. Sequences of the viral termini were determined by artificial polyadenylation (30end) and/or RACE (50end) methodology using a GeneRacer kit (Invitrogen, USA). Comparisons with sequences available in the NCBI/Gen- Bank database were performed using the BLAST [2] and CDD [20] resources. Phylogenetic analysis with bootstra- ping consisting of 1,000 pseudoreplicates was performed with ClustalX software [17] using the neighbor-joining algorithm, and trees were visualized with the Dendroscope program [15]. The genome of this virus, provisionally denoted grape- A B vine leafroll-associated Carnelian virus (GLRaCV), con- sists of 13,625 nt (Fig. 2) and closely resembles that of Fig. 1 a Mild leafroll symptoms induced by GLRaCV on indicator several recently sequenced ‘‘short’’ ampeloviruses: plum Vitis vinifera cv. Cabernet Franc. b Electrophoretic patterns of bark necrosis stem pitting-associated virus (PBNSPaV) [3], dsRNAs extracted from cv. Carnelian (lane 1) compared to dsRNAs extracted from grapevine infected with grapevine rupestris stem GLRaV-Pr and -De [18, 19], and pineapple mealybug wilt- pitting-associated virus (lane 2) and from GLRaV-2-infected Nico- associated viruses 1 and 3 (PMWaV-1 and PMWaV-3) tiana benthamiana (lane 3) [24, 29]. The GLRaCV genome starts with an AU-rich (56%) GRSPaV), the full genome-size molecule was estimated to 214-nt-long non-coding region (50NCR). The first ORF be ca. 13.5 kbp (Fig. 1b). (ORF1a) extends for 2,288 codons and codes for a repli- Initial molecular characterization of this virus was car- cation-associated polyprotein with an estimated molecular ried out on a portion of the HSP70-homologue (HSP70h) mass of 254.7 kDa (p255) containing hallmark domains of cistron between motifs P1 and P2 that was amplified using (in the 50-to-30 direction) papain-like protease [12], viral universal degenerate primers for closterovirids [30]. Initial methyltransferase (MTR, pfam 01660) [26], and viral sequence data showed that this virus was fairly different helicase superfamily 1 (HEL, pfam 01443) [11]. Addi- from other LRD-associated viruses (ca. 70% identity at the tionally, p255 contains a conserved AlkB domain (2OG- amino acid level) and other members of the genus Fe(II) oxygenase superfamily; pfam 03171) [5], which is Ampelovirus. located between MTR and HEL and was reported previ- Further sequence data were generated by random- ously to be present in some other ampeloviruses, i.e. little primed reverse transcription of purified dsRNA prepara- cherry virus 2, GLRaV-3 [5] and GLRaV-Pr [19]. Func- tions and cloning into pGEM-T Easy plasmid (Promega tional domains identified in p255 showed considerable Corporation, USA). Selected plasmids were sequenced at conservation of amino acids with related products encoded the UC Davis and/or Mississippi State University DNA by GLRaV-Pr and -9. Helicase was the most conserved Sequencing Facility. Sequence data were assembled and domain, sharing ca. 70% aa identity with GLRaV-Pr and - analyzed using DNAStar (Lasergene) software. The 9, whereas about 60% of the residues of MTR and AlkB sequences that were generated were used to design virus- are identical to those of these closely related viruses specific primers in order to progress towards sequencing (Table 1). As in other closterovirids, conserved functional the whole genome. Each genomic region was verified by domains were separated by regions with variable aa

p5 PRO MTR AlkB HEL p60 5’ 3’ RdRp HSP70h CP p23

1 2,000 4,000 6,000 8,000 10,000 12,000 13,625

Fig. 2 Diagrammatic representation of the GLRaCV genome to p5 hydrophobic 5 K protein; HSP70h heat shock 70 protein scale. Boxes depict putative ORFs. PRO protease; MTR methyltrans- homologue; p60 60 K protein; CP coat protein; p23 23 K protein ferase; HEL helicase; RdRp RNA-dependent RNA polymerase; with unknown function

123 A new ampelovirus associated with grapevine leafroll disease 1873

Table 1 Percent nucleotide (nt) or amino acid (aa) identity between genomes/genome products of GLRaCV and closely related grapevine leafroll-associated viruses/isolates of the subgroup I in the genus Ampelovirus Virus % nt identity % aa identity Genome 50UTR 30UTR MTR HEL ORF1b p5 HSP70h p60 CP p23

GLRaV-4 66* 65 78 52* GLRaV-5* 63* 66 63 77 56 GLRaV-6 66* GLRaV-9* 60* 53* 87* 55 69 77 73 67 64 78 56 GLRaV-Pr 60 58 76 58 71 76 69 69 64 77 50 GLRaV-De 63* 66 63 76 62* * Partial sequences content and length, reducing the overall identity between 125, 172 and 213, respectively. This protein shares 76–78% GLRaCV and GLRaV-Pr (the closest fully sequenced aa identity with orthologs of GLRaV-5 and related viruses, virus) to ca. 45%. ORF1a terminates with the sequence including the recently reported GLRaV-Pr and -De [18, 19]. guuUAAca (stop codon in capital letters), which is iden- Values of amino acid content conservation with CPs tical to the corresponding region in PMWaV-1 [24] and is of PBNSPaV, PMWaV-1 and -3 are 27, 55 and 62%, presumably involved in a ?1 ribosomal frameshift phe- respectively. nomenon. ORF1b overlaps ORF1a for 8 nucleotides and The 30-end-proximal ORF6 codes for a putative p23 codes for a polypeptide with a deduced molecular mass protein with 50–56% identity with the corresponding gen- of 59.1 kDa containing the conserved sequence motifs ome products of related viruses. Direct comparison of p23 of a viral RNA-dependent RNA polymerase (RdRp) of with the viral CP showed low overall identity (16%) scat- the ‘‘alphavirus-like’’ viruses [16]. This protein shares tered randomly throughout the protein. The absence of sig- 76–77% identity with orthologs of the most closely related nificant amino acid conservation between the two proteins, ampeloviruses (GLRaV-9 and –Pr; Table 1). Identities as well as the lack of the ‘‘closter_coat motif’’ (pfam 01785) with other short ampeloviruses were much lower (i.e. 37% suggests that p23 may not be a paralog of the viral coat with PBNSPaV and 53% with PMWaV-1). protein and may have a completely different function. The After a 4-nt-long intergenic region, the coding part of genome terminates with a 131-nt-long 30 NCR that is 87 and the genome continues with a small ORF (ORF2) encoding 76% identical to the corresponding regions of GLRaV-9 and a 46-aa-long hydrophobic protein (p5) with membrane- GLRaV-Pr, respectively. This protein shares 30 and 37% aa binding properties. It shared the highest level of amino acid identity with PMVaV-1 and -3, and\15% with PBNSPaV. sequence identity (73%) with the hydrophobic protein In phylogenetic analysis, performed on several phylo- encoded by the GLRaV-9 genome. genetically relevant gene products (i.e. RdRp, HSP70h, The 58-kDa polypeptide encoded by ORF3 contains the CP), GLRaCV always grouped with members of subgroup conserved motifs of cellular chaperones and shares \70% I in the genus Ampelovirus. Nevertheless, independent of identical amino acids with HSP70 homologues encoded by the gene used in analysis, GLRaCV appeared to be the the GLRaV-4, -5, -6, -9, -Pr and -De genomes (Table 1). most distinct member of the ‘‘GLRaV-4 clade’’, as it was Identities with other related viruses were in the range of 43 consistently placed slightly apart from the rest of these (PBNSPaV) to 59% (PMWaV-1). ORF4 partially overlaps viruses (Fig. 3). This topology was also supported by with the previous cistron and extends for 1,620 nt. It Bayesian inference and maximum-likelihood methods encodes a 539-aa-long protein related to the ca. 60-kDa applied in MrBayes [14] and PhyML [13] software, proteins of other closterovirids with a role in virion/tail respectively (not shown). assembly/virus movement [8]. This ORF has fewer than Based on our results, the virus identified in the LRD- 65% of its amino acids in common with the corresponding affected grapevine plant of cv. Carnelian appears to be a products of closely related ‘‘GLRaV-4-like’’ viruses/vari- member of a novel viral species in the genus Ampelovirus. ants, and only 48–49% with PMWaV-1 and -3. It induced mild leafroll symptomatology in the original ORF5 is separated by 69 nt from the previous cistron and plant as well as in the leafroll-specific indicator host. Virus encodes a viral coat protein with a predicted molecular mass was not detected by RT–PCR using a panel of primers of 29.2 kDa. Serine, arginine and aspartic acid residues specific for GLRaV-1 to -7 and -9 that are routinely used reported to be conserved in coat proteins (CP) of filamentous for diagnostic purposes at the Foundation Plant Services and rod-shaped phytoviruses [7] were identified at positions laboratory at UC Davis. 123 1874 N. Abou Ghanem-Sabanadzovic et al.

Outgroup 702 LChV-1 GLRaV-7 Unassigned 987 1000 LIYV TICV 1000 CYSDV 840 BYVaV Gen. Crinivirus 1000 858 PYVV SPCSV

811 CNFV 991 CYLV MV-1 RMoV Gen. Closterovirus 1000 CTV BYSV 996 BYV GLRaV-2

708 LChV-2 862 PMWaV-2 Subgroup

GLRaV3 II 978 GLRaV-1 981 PBNSPaV 1000 PMWaV-3

989 PMWaV-1

1000 GLRaV-Pr Ampelovirus Subgroup 1000 GLRaV-De I GLRaV-9 Gen. 838 GLRaV-5 981 0.05 GLRaV-4 GLRaCV

Fig. 3 Phylogenetic tree constructed using HSP70h amino acid AM494935), Pr (GLRaV-Pr, NC_011702), grapevine leafroll-associ- sequences of members of some definitive and tentative species in the ated Carnelian virus (GLRaCV, FJ907331), little cherry virus 1 family Closteroviridae. Bootstrap values are indicated on the nodes. (LChV-1, NC_001836), little cherry virus 2 (LChV-2, NC_005065), Sequences used to construct the tree and their accession numbers are lettuce infectious yellows virus (LIYV, NC_003618), mint virus-1 as follows: beet yellows virus (BYV, NC_001598), beet yellow stunt (MV-1, NC_006944), pineapple mealybug wilt-associated virus 1 virus (BYSV, U51931), blackberry yellow vein associated virus (PMWaV-1, NC_010178), pineapple mealybug wilt-associated virus (BYVaV, NC_006963), carnation necrotic fleck virus (CNFV, 2 (PMWaV-2, AF283103), pineapple mealybug wilt-associated virus EU884443), carrot yellow leaf virus (CYLV, NC_013007), citrus 3 (PMWaV-3, DQ399259), plum bark necrosis stem pitting associ- tristeza virus (CTV, NC_001661), cucurbit yellow stunting disorder ated virus (PBNSPaV, NC_009992), potato yellow vein virus (PYVV, virus (CYSDV, NC_004810), grapevine leafroll-associated virus 1 NC_006063), raspberry leaf mottle virus (RLMoV, NC_008585), (GLRaV-1, AF195822), 2 (GLRaV-2, NC_007448), 3 (GLRaV-3, sweet potato chlorotic stunt virus (SPCSV, NC_004124) and tomato NC_004667), 4 (GLRaV-4, AM162280), 5 (GLRaV-5, AF233934), 7 infectious chlorosis virus (TICV, NC_013259). Outgroup: putative (GLRaV-7, Y15987), 9 (GLRaV-9, AY297819), De (GLRaV-De, heat shock protein 70 of Arabidopsis thaliana (AAN71949)

Complete sequencing of this virus showed a genomic of the GLRaCV genome differs by more than 25% from organization similar to those of GLRaV-Pr [19], GLRaV-9 orthologs in the closely related GLRaV-5, -9, -De and -Pr, as [4], PMWaV-1 [24], PMWaV-3 [29] and PBNSPaV [3], the well as with GLRaV-4 and -6 (unpublished data). Indepen- genomes of which are completely or almost completely dent of the genome product used for analysis, identities with known, and presumably to a number of other, partially other approved or putative ‘‘short’’ ampeloviruses (i.e. characterized ‘‘short GLRaVs’’ (i.e. GLRaV-4, -5, -6, -De PMWaV-1, -3, PBNSPaV) were at least 10–15% lower than and -WC15) [1, 10, 18, 25]. However, in-depth analysis values with ‘‘GLRaV-4-like viruses’’. The replication- showed that the isolate from cv. Carnelian shares rather associated polyprotein (p255) and p23 (encoded by ORF6) limited amino acid identity with sequenced members of this appear to be less conserved than their orthologs in GLRaV-9 subgroup of ampeloviruses. With the exception of the viral and -Pr (Table 1). In the case of p255, the overall polypro- CP, which shares 76–78% common residues with its closest tein shares only 44 and 48% aa identity with GLRaV-9 and relatives, the amino acid content of other putative products GLRaV-Pr, respectively, due to the poorly conserved

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